The present invention relates to polypeptides having substituted one or more amino acid residues to the polypeptide and/or having coupled polymeric molecules on the surface of the 3-dimensional structure of the polypeptide, a method for preparing modified polypeptides of the invention, the use of the modified polypeptides for reducing immunogenicity and allergenicity, and compositions comprising the polypeptide.
The use of polypeptides, including enzymes, in the circulatory system to obtain a particular physiological effect is well-known in the medical arts. Further, within the arts of industrial applications, such as laundry washing, textile bleaching, personal care, contact lens cleaning, and food and feed preparation enzymes are used as a functional ingredient. One of the important differences between pharmaceutical and industrial application is that for industrial applications the polypeptides (often enzymes) are not intended to enter into the circulatory system of the body.
Certain polypeptides and enzymes have an unsatisfactory stability and may under certain circumstancesxe2x80x94dependent on the way of challengexe2x80x94cause an immune response, typically an IgG and/or IgE response.
It is today generally recognized that the stability of polypeptides is improved and the immune response is reduced when polypeptides, such as enzymes, are coupled to polymeric molecules. It is believed that the reduced immune response is a result of the shielding of (the) epitope(s) on the surface of the polypeptide responsible for the immune response leading to antibody formation by the coupled polymeric molecules.
Techniques for conjugating polymeric molecules to polypeptides are well-known in the art.
One of the first suitable commercial techniques was described in the early 1970""s and disclosed in e.g. U.S. Pat. No. 4,179,337. This patent concerns non-immunogenic polypeptides, such as enzymes and peptide hormones coupled to polyethylene glycol (PEG) or polypropylene glycol (PPG). At least 15% of the polypeptides"" physiological activity is maintained.
GB patent no. 1,183,257 (Crook et al.) describes chemistry for conjugation of enzymes to polysaccharides via a triazine ring.
Further, techniques for maintaining the enzymatic activity of enzyme-polymer conjugates are also known in the art.
WO 93/15189 (Veronese et al.) concerns a method for maintaining the activity in polyethylene glycol-modified proteolytic enzymes by linking the proteolytic enzyme to a macromolecularized inhibitor. The conjugates are intended for medical applications.
It has been found that the attachment of polymeric molecules to a polypeptide often has the effect of reducing the activity of the polypeptide by interfering with the interaction between the polypeptide and its substrate. EP 183 503 (Beecham Group PLC) discloses a development of the above concept by providing conjugates comprising pharmaceutically useful proteins linked to at least one water-soluble polymer by means of a reversible linking group.
EP 471,125 (Kanebo) discloses skin care products comprising a parent protease (Bacillus protease with the trade name Esperase(copyright)) coupled to polysaccharides through a triazine ring to improve the thermal and preservation stability. The coupling technique used is also described in the above mentioned GB patent no. 1,183,257 (Crook et al.).
JP 3083908 describes a skin cosmetic material which contains a transglutaminase from guinea pig liver modified with one or more water-soluble substances such as PEG, starch, cellulose etc. The modification is performed by activating the polymeric molecules and coupling them to the enzyme. The composition is stated to be mild to the skin.
WO 98/35026 (Novo Nordisk A/S) describes polypeptide-polymer conjugates having added and/or removed one or more attachment groups for coupling polymeric molecules on the surface of the polypeptide structure. The conjugates have reduced immunogenicity and allergenicity.
It is the object of the present invention to provide improved polypeptides suitable for industrial and pharmaceutical applications.
The term xe2x80x9cimproved polypeptidesxe2x80x9d means in the context of the present invention polypeptides having a reduced immune response in humans and animals. As will be described further below the immune response is dependent on the way of challenge. The present inventors have found that polypeptides, such as enzymes, may be made less immunogenic and/or allergenic by substituting one or more amino acid residues on the surface of the polypeptide with other amino acid residues and/or by coupling polymeric molecules on the surface of the enzyme in the vicinity of a bound ligand of the enzyme e.g. a metal ion substantially without affecting the enzymatic activity.
When introducing pharmaceutical polypeptide directly into the circulatory system (i.e. bloodstream) the potential risk is an immunogenic response in the form of mainly IgG, IgA and/or IgM antibodies. In contrast hereto, industrial polypeptides, such as enzymes used as a functional ingredient in e.g. detergents, are not intended to enter the circulatory system. The potential risk in connection with industrial polypeptides is inhalation causing an allergenic response in the form of mainly IgE antibody formation.
Therefore, in connection with industrial polypeptides the potential risk is respiratory allergenicity caused by inhalation, intratracheal and intranasal presentation of polypeptides.
The main potential risk of pharmaceutical polypeptides is immunogenicity caused by intradermal, intravenous or subcuaneous presentation of the polypeptide.
The term xe2x80x9cimmunogenicityxe2x80x9d used in connection with the present invention may be referred to as allergic contact dermatitis in a clinical setting and is a cell mediated delayed immune response to chemicals that contact and penetrate the skin. This cell mediated reaction is also termed delayed contact hypersensitivity (type IV reaction according to Gell and Combs classification of immune mechanisms in tissue damage).
The term xe2x80x9callergenicityxe2x80x9d or xe2x80x9crespiratory allergenicityxe2x80x9d is initially an immediate anaphylactic reaction (type I antibody-mediated reaction according to Gell and Combs) following inhalation of e.g. polypeptides.
According to the present invention it is possible to provide polypeptides with a reduced immune response, which has a substantially retained residual activity.
The allergic and the immunogenic response are in one term, at least in the context of the present invention called the xe2x80x9cimmune responsexe2x80x9d.
In the first aspect the invention relates to a polypeptide with reduced immune response, having one or more amino acid residues modified, wherein the Cxcex1-atoms of the amino acid residues are located less than 15 xc3x85 from the ligand bound to the polypeptide.
The reduced immune response is preferably reduced allergenicity.
The modification of the polypeptide is conducted by substituting one or more amino acid residues in the parent polypeptide with other amino acid residues to the polypeptide, and/or by selecting variants from a diverse library of variants of the parent polypeptide and/or by coupling a polymeric molecule to the surface of the parent polypeptide.
The term xe2x80x9cparent polypeptidexe2x80x9d refers to the polypeptide to be modified by coupling to polymeric molecules or by substituting amino acid residues. The parent polypeptide may be a naturally-occurring (or wild-type) polypeptide or may be a variant thereof prepared by any suitable means. For instance, the parent polypeptide may be a variant of a naturally-occurring polypeptide which has been modified by substitution, deletion or truncation of one or more amino acid residues or by addition or insertion of one or more amino acid residues to the amino acid sequence of a naturally-occurring polypeptide.
A xe2x80x9csuitable attachment groupxe2x80x9d means in the context of the present invention any amino acid residue group on the surface of the polypeptide capable of coupling to the polymeric molecule in question.
Preferred attachment groups are amino groups of Lysine residues and the N-terminal amino group. Polymeric molecules may also be coupled to the carboxylic acid groups (xe2x88x92COOH) of amino acid residues in the polypeptide chain located on the surface. Carboxylic acid attachment groups may be the carboxylic acid group of Aspartate or Glutamate and the C-terminal COOH-group. Another attachment group is SH-groups in Cysteine.
An xe2x80x9cactive sitexe2x80x9d means any amino acid residues and/or molecules which are known to be essential for the performance of the polypeptide, such as catalytic activity, e.g. the catalytic triad residues, Histidine, Aspartate and Serine in Serine proteases, or e.g. the heme group and the distal and proximal Histidines in a peroxidase such as the Arthromyces ramosus peroxidase.
A xe2x80x9cligandxe2x80x9d, means in the context of the present invention a metalor, a metal ion or a cofactor.
In the context of the present invention xe2x80x9cmodification of amino acid residuesxe2x80x9d means that amino acid residues are substituted with other amino acid residues and/or a polymeric molecule is coupled to the amino acid residue. The polypeptide of the present invention may according to the invention be modified by substitution alone, by coupling of a polymeric molecule alone or by a combination of substitution and coupling.
In the context of the present invention xe2x80x9clocatedxe2x80x9d means the shortest distance from any atom in the ligand to the relevant C-atom in the amino acid residue.
Furthermore, in the context of the present invention, e.g. xe2x80x9cR250Kxe2x80x9d means that the amino acid Arginine in position 250 of the polypeptide has been substituted with the amino acid Lysine according to the one-letter-code of amino acids.
In the second aspect the invention relates to a method for preparing polypeptides with reduced immune response comprising the steps of:
a) identifying amino acid residues located on the surface of the 3-dimensional structure of the parent polypeptide in question,
b) selecting target amino acid residues on the surface of the 3-dimensional structure of the parent polypeptide to be modified,
c) substituting one or more amino acid residues selected in step
b) with other amino acid residue, and/or
d) coupling polymeric molecules to the amino acid residues in step b) and/or step c).
The invention also relates to the use of a modified polypeptide of the invention and the method of the invention for reducing the immunogenicity of pharmaceuticals and reducing the allergenicity of industrial products.
Finally the invention relates to compositions comprising a modified polypeptide of the invention and further ingredients used in industrial products or pharmaceuticals.